High-throughput screening assay for PARP-HPF1 interaction inhibitors to affect DNA damage repair

ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents.

www.nature.com/scientificreports/HPF1, PARP1/2 predominantly modify serine residues 8,9 .As an alternative strategy to already developed PARP inhibitors, it could be possible to prevent binding of HPF1 to PARP1/2 to inhibit serine ADP-ribosylation in DNA repair.HPF1 initiates PARylation on specific serine residues of histone tails subsequently leading to remodeling of the nucleosome with help of other factors such as ALC1 recognizing the modification 24 .As HPF1 shares a joint active site with PARP1/2 and plays a key role in increasing enzymatic activities, the inhibition of the interaction between HPF1-PARP would reduce histone PARylation upon DNA damage as single agent or possibly in combination with catalytic PARP inhibitors or DNA damaging agents.In the crystal structure, HPF1 and PARP2 interaction is mediated by a 15 amino acid long helix (261-284) of HPF1 packing against the PARP2 catalytic domain and NAD + binding cleft 12 (Fig. 1a).Through this, HPF1 and PARP2 collectively form a joint active site.The last two residues of PARP2 C-terminus interact at the back of HPF1 C-terminal domain and the overall interface buried is 1328.6Å 225 .
In order to generate a signal that can be used in high-throughput mode to discover protein-protein interaction inhibitors as potential starting points for drug development, we decided to label the proteins with fluorophores to develop a FRET assay.The helical autoinhibitory domain would interfere in PARP-HPF1 interaction and hence we expressed fusion proteins where mCerulean (CFP) is attached to the N-terminus of the PARP ART domains.The mCitrine (YFP) is attached to the N-terminus of HPF1 as then the tags would not interfere with the complex formation based on the structure.When there is an interaction between labeled HPF1 and PARP1/2 then YFP and CFP come in close proximity and upon excitation of CFP there is an energy transfer from CFP to YFP resulting in FRET signal (Fig. 1b).Protein-protein interaction inhibitor would result in a loss of the FRET signal.We optimized the assay for screening of inhibitors of PARP2-HPF1 interaction and showed that the homogeneous assay is robust and can be run on a 384-well microplates in high-throughput screening campaigns.During the assay validation, we discovered two compounds Dimethylacrylshikonin and Alkannin as inhibitors of the protein-protein interaction.

Assay setup and FRET measurement
To measure the interaction between PARP1/2 and HPF1 and to develop a high throughput method for inhibitor screening we used a FRET-based approach.We cloned constructs of PARP1 and PARP2 ART domains fused with CFP at the N-terminus and HPF1 fused with YFP at the N-terminus.Purified CFP-PARP1/2 and YFP-HPF1 were mixed, and FRET was measured by excitation of CFP at 410 nm wavelength and measuring YFP fluorescence intensity (527 nm) and CFP fluorescence intensity (477 nm).When CFP and YFP interact, the CFP emission at 477 nm decreases while YFP emission at 527 nm increases.The ratio of emission intensities of 527 nm and 477 nm, rFRET, was used throughout the work as a measure of binding interaction when comparing it with controls.As the yields for produced CFP-PARP2 were higher than those for CFP-PARP1 (25 mg/l and 10 mg/l, respectively), we decided to use CFP-PARP2 for optimization and for screening.For the interaction between HPF1 and PARP2, buffer conditions i.e., pH, buffering reagent, buffer and salt concentration, additives were optimized to get a robust and reproducible signal (Fig. 2).
First the effect of pH on rFRET (5 µM YFP-HPF1 and 1 µM CFP-PARP2) was tested using 10 mM Bis-Tris-Propane (BTP), which has pH ranges from 6.0 to 9.5 (Fig. 2a).At lower pH at 6.0, YFP-HPF1 (5 µM) and CFP-PARP2 (1 µM) FRET pairs showed maximum signal but it was evident that at pH at 6.0 and 6.5 proteins were aggregating in the well and this was interpreted as the source of the higher rFRET signal.Increasing pH resulted in diminishing signal, while at neutral pH 7.0 the rFRET signal was present and there were no signs of protein aggregation in the well.Therefore, we decided to further optimize the buffer conditions at pH 7.0, which is also close to physiological pH and therefore the ionization state of the potential inhibitors will be reasonable.After optimizing pH, 10 mM buffering reagents at pH 7.0 were tested for the FRET pair and since the highest signal was observed with BTP we decided to continue with it (Fig. 2b).Increasing concentrations of the buffering agent, however, decreased the signal (Fig. 2c).This could be an indication of sensitivity of the system to increasing salt concentrations and indeed, when we tested NaCl concentrations from no salt to 1 M we observed that even low salt concentration of 50 mM drastically reduced the rFRET signal (Fig. 2d).We therefore decided to use no salt in the buffer and tested the effect of Polyethylene Glycol 20,000 (PEG 20k) as a crowding agent.PEG has shown to enhance the rFRET signal and help in blocking adhesion the biomolecules to plastic walls 26 .Increasing PEG concentrations resulted in an increase in the rFRET signal (Fig. 2e).At higher PEG concentration rFRET signal www.nature.com/scientificreports/increased very rapidly to unusually high rFRET values based on our previous experience 27,28 and we interpreted this to be due to unspecific interactions.We decided therefore to add only a small amount of 3% (w/v) PEG 20k to the FRET buffer.Triton X-100 was found to slightly increase the signal and as it could help in the assay setup including the meniscus formation in the experimental plate, we also added 0.01% (w/v) to the final FRET buffer (Fig. 2f).Based on the optimization, 10 mM BTP, 0.01% tritonX-100, 0.5 mM TCEP, 3% PEG 20k, pH 7.0, was selected as an optimal FRET buffer.We next wanted to test the effect of HPF1-PARP2 stoichiometry and whether we would be able to reduce the proteins concentration while maintaining a good enough signal for screening.Increase in the relative HPF1 concentration slightly increased the rFRET signal systematically as more complex would be present in the mixture (Fig. 2g).The difference was however small and to save protein and to keep the sensitivity of the assay reasonable we made a compromise and decided to use 400 nM CFP-PARP2 and 800 nM YFP-HPF1 for further studies.

Signal and protein stability
In the initial part of optimization procedure, we studied the stability of the rFRET signal over time (Fig. 3a).We observed that the rFRET signal was increasing with time indicating non-steady state conditions that could be a result of instability of the proteins.This increase in signal over time would be a problem also in the context of a larger screening campaign as the dispensing and readouts need to be precisely timed.As the preparation of PARP1 and PARP2 without the autoinhibitory helical domain has been challenging we suspected that the protein constructs would not be stable in the used low salt buffer and measured the thermal stability using differential scanning fluorimetry (nanoDSF).PARP2 started unfolding at very low temperatures (Tm 25.6 ± 0.15 °C) with a not very well defined transition (Fig. 3b).www.nature.com/scientificreports/ The increasing rFRET signal and nanoDSF data, collectively suggested that the protein was not stable at room temperature and was aggregating with time potentially bringing the fluorophores to proximity.Buffer additives such as glycine, guanidium hydrochloride, polyoxyethylene lauryl ether (Brij-35) were tested to stabilize the CFP-PARP2 protein, but additives did not help in stabilizing the protein in FRET buffer (data not shown) 29,30 .To stabilize the PARP2 fragment, fivefold excess of PARP catalytic inhibitor Olaparib was added in the FRET buffer.Addition of Olaparib resulted in a sharp unfolding transition for PARP2 with higher Tm value (44.6 ± 0.07 °C) by making the protein stable at room temperature (Fig. 3b).For the fusion protein 2 transitions were expected as it contains 2 independent domains with different Tms.The thermal melting was run up to 60 °C and Tm for CFP is 80 °C, so a single transition was observed for PARP2 ART domain only.In the presence of Olaparib, CFP-PARP2 and YFP-HPF1 interaction was stable in the FRET assay and simultaneously confirmed that Olaparib was not interfering in the PARP2-HPF1 interaction and could be used in the interaction assay (Fig. 3c).
In the presence of Olaparib, 400 nM CFP-PARP2 and 800 nM YFP-HPF1 were mixed resulting in a stable signal within an experiment, but the rFRET values were not reproducible in different experiments.This could indicate variation in complex formation between experiments depending on the order of addition and salt concentration that comes from the independently purified proteins.After trial and error, FRET pair proteins were mixed first in higher concentration (25 µM CFP-PARP2, 50 µM YFP-HPF1) in the buffer containing excess Olaparib (100 µM) in a so-called intermediate mixture.Final FRET mixture with 400 nM CFP-PARP2 and 800 nM YFP-HPF1 was then prepared by diluting the intermediate mixture in FRET buffer.Mixing of the proteins in high concentration allows formation of complex in saturating amounts before the dilution to the lower assay conditions.This procedure allowed us to get a stable, reproducible, and higher rFRET signal in comparison to a direct mixing to the final FRET conditions (Fig. 3d).

Signal verification
In the optimization phase we used rFRET as a direct readout for the interaction and secondary information like protein aggregation to judge whether the signal resulted from the interaction between the proteins.To test that the interaction was not with a fluorescent tag we carried out a control titration experiment (Fig. S3).To further confirm the specific interaction between PARP2 and HPF1, we next tested if unlabeled HPF1 would compete with the FRET pair and expected that the competition would result in the loss of rFRET signal.Increasing concentration of unlabeled HPF1 (35 nM-500 mM) in the reaction indeed decreased the rFRET signal in a concentration dependent manner, but it should be noted here that there is salt added also to the mixture coming from the unlabeled HPF1 protein stock (Fig. 4a).To rule out the effect of added salt, we repeated the experiment in the absence of HPF1 but using equivalent salt concentrations (25 nM-350 mM) which resulted in higher IC 50 (pIC 50 : 4.2 ± 0.01, IC 50 : 64 µM) as compared to the competition by HPF1 (pIC 50 : 5.8 ± 0.04, IC 50 : 1.5 µM).The 1.6-log unit difference in the apparent IC 50 values clearly indicates that unlabeled HPF1 competes with the FRET pair (Fig. 4a).To further strengthen the analysis and to confirm the competition we also exchanged the HPF1 buffer.The desalted unlabeled HPF1 also showed concentration dependent lowering (pIC 50 : 4.62, IC 50 : 24.0 µM) of the rFRET signal (Fig. 4b).We however observed aggregation of HPF1 in no salt condition at high concentrations and therefore the active unlabeled HPF1 protein concentration in the reaction was likely lower than the calculated total concentration while the competition was still evident.These results confirmed that unlabeled HPF1 (without salt) competed with the CFP-PARP2 and YFP-HPF1 FRET pair and validated the interaction between HPF1 and PARP2 in the FRET reaction.
In addition to the FRET pair competition with unlabeled HPF1, we also measured dynamic light scattering (DLS) for the CFP-PARP2 (0.5 mg/ml), YFP-HPF1 (1.0 mg/ml), and CPF-PARP2/YFP-HPF1 complex (Fig. S4a,c,d respectively).In DLS, even at the high protein concentration, scattering starts above 30 °C temperature confirming that there is no aggregation at room temperature (25 °C) and the FRET signal is the result of PARP2-HPF1 interaction.www.nature.com/scientificreports/

Dissociation constant for PARP2-HPF1 interaction
The binding affinity of PARP2-HPF1 was measured using FRET according to the method described by Song et al. 31 .An increasing concentration of YFP-HPF1 (250 nM to 10.5 µM) was mixed with a constant concentration of CFP-PARP2 (400 nM) to saturate the CFP with YFP signal.Here we did not consider the effect of salt as ionic strength does not impact the interaction at high protein concentrations.Based on the raw fluorescence data, the FRET emission (Em FRET ) was calculated, and the saturation curve showed Kd of 2.0 ± 0.18 µM (Fig. 4c).This is in line with the values and estimates from the literature 1.5 µM for HPF1-PARP1(ART only) 13 , while the Kd of 3 µM was reported for full length wild type PARP2 32 .The Kd value for HPF1-PARP2 (ART) indicates that in the preparation of the FRET mixture the complex is formed in the intermediate mixing (25 µM CFP-PARP2, 50 µM YFP-HPF1) and this is then partially dissociated when diluted to the final FRET buffer (400 nM CFP-PARP2 and 800 nM YFP-HPF1).

Signal validation for screening
Once the CFP-PARP2 and YFP-HPF1 interaction was verified, the rFRET signal performance was validated by repeating the experiments on different days using different plates.The experiments were performed in 10 µl and 20 µl reaction volumes in 384-well plates, which contained 176 replicates of FRET pair (positive control) and 176 replicates of FRET pair with 1 M NaCl (negative control) (Fig. 5).The Zʹ-factors for 10 µl and 20 µl FRET reaction volumes were determined in a range of 0.85 to 0.92 and 0.75 to 0.88, respectively (Table S1 and S2).The Z' factor value above 0.5 indicates the separation between negative and positive control is large, and assay with Z' value above 0.5 is considered as excellent 33 .The statistical analysis suggests that even in a low assay volume of only 10 µl the quality of the signal was consistent, and the assay can be used for the screening of inhibitors.

Validatory screening
To validate the assay for inhibitor screening, Targetmol inhibitor compound library (1832 compounds), which contains biologically active compounds, was screened (Fig. 5c).For the screening, 10 nl compounds (10 µM concentration in reaction) and 10 µl FRET pair protein mixture (400 nM CFP-PARP2 and 800 nM YFP-HPF1) was dispensed and measured.In the presence of inhibitors, an additional excitation wavelength 430 nm was also used for the FRET measurement to rule out spurious effects due to compound fluorescence.This is based on the idea that for true protein-protein interaction inhibitors the inhibition at different excitation wavelengths is expected to be similar.On the other hand, artifacts due to small molecules fluorescence are expected to have different effects at different excitation wavelengths since they usually have sharper emission and excitation spectra.To analyze inhibition data, multiple filters were applied to rule out non-specific FRET signal coming from intrinsic fluorescence of individual compounds.Raw fluorescence signal with more than 20% fluorescence value (> 1.2* fluorescence at 527 nm; < 1.2*fluorescence at 477 nm) from the positive control FRET pair fluorescence signal, were considered as outlier and were not included for further analysis (Table S3).The % rFRET activity was calculated for each compound based on the controls.The hit limit for library compounds was set to 70.25% activity (5* standard deviations from mean) (Table S3).In the screening, a total of 3 compounds Dimethylacrylshikonin (CAS no.24502-79-2), Alkannin (CAS no.517-88-4), Crystal violet (CAS no.548-62-9) were identified as hits for PARP2-HPF1 interaction (Fig S2 d-f).

Hit validation
To validate the hit compounds from CFP-PARP2 and YFP-HPF1 FRET pair screening, the binding of the compounds (Dimethylacrylshikonin, Alkannin, Crystal violet) with PARP2 and HPF1 were measured using nanoDSF (Fig. S2).When compound binds to the protein it results in the increase in the melting temperature of the protein.
The Tm values for PARP2 and HPF1 in absence of hit compound were 44.2 ± 0.10 °C and 46.8 ± 0.02 °C, respectively.In the presence of hit compounds Dimethylacrylshikonin, Alkannin, and Crystal violet, the Tm value of PARP2 were measured as 46.0 ± 0.4 °C, 46.0 ± 0.2 °C, and 40.0 ± 0.5 °C, respectively (Fig. S2a-c).The increase in Tm in presence of Dimethylacrylshikonin and Alkannin, suggested their binding to PARP2 ART domain, but Crystal violet destabilizes the protein.The compounds did not cause a thermal shift for HPF1.www.nature.com/scientificreports/HPF1 has been crystallized with ART domain of PARP1 (PDB id: 6M3I) 13 and PARP2 (PDB id: 6TX3) 12 .The ART domains of both PARP1 and PARP2 share a similar tertiary structure and interact with HPF1 approximately with the same interface and form a joint active site (Fig. S1).To study also the interaction between PARP1 ART domain and HPF1, CFP-PARP1 was mixed with YFP-HPF1 in 1:2 concentration ratio in optimized buffer for PARP2-HPF1 FRET pair (Fig. 6a).The FRET was measured using the same parameters as used for CFP-PARP2 and YFP-HPF1 FRET pair and a stable FRET signal was observed (Fig. 6b), which is not due to aggregation of CPF-PARP1/YFP-HPF1 complex (Fig. S4e).The assay thus allowed us to measure potencies of inhibitors also for the PARP1-HPF1 interaction.

Inhibitory effect of compounds on PARP1/2 activity
Activities of full-length PARP1 or PARP2 were measured in presence of Dimethylacrylshikonin and Alkannin to study their inhibitory effect on PARP1/2 activity (Fig. 8).In presence of HPF1, PARP1/2 activity increases and treatment with hydroxylamine selectively removes PAR chains linked to Asp and Glu but do not affect Ser ADP-ribosylation 34,35 and we confirmed this as a control (Fig. S5).The addition of 100 µM inhibitor compounds Dimethylacrylshikonin and Alkannin to the PARP activity reaction resulted in a decrease in PAR smear and increase of unmodified PARP1 (Fig. 8a, lane 6, 7 and 12, 13) and PARP2 (Fig. 8b, lane 6, 7, and 13, 14).The higher concentration of inhibitor compounds (1 mM) drastically reduced the amount of PAR smear and most PARP1 (Fig. 8a, lane 8, 14) and PARP2 (Fig. 8b, lane 8, 15) remained unmodified.The reduction in PARP1/2 modification confirmed that compounds interfere with PARP-HPF1 interaction in the context of full-length proteins and decrease the PARP activity resulting in the decrease in PAR formation.

Discussion
Current PARP inhibitors target the catalytic site and compete with NAD + .In the context of DNA damage the main form of ADP-ribosylation is on serine residues and dependent on HPF1.Interfering with the PARP-HPF1 interaction provides an alternative and unique way to inhibit PARP activity 8,12,13 .Protein-protein interactions are known to be challenging to target and so far there are no inhibitors reported for the PARP-HPF1 interaction.In this study, we have developed a FRET-based high-throughput screening assay that allows direct monitoring of HPF1-PARP interaction.The assay can be used for screening of small inhibitor molecules against PARP-HPF1 interaction.The proteins are expressed as fusion proteins with fluorescent proteins and when PARP interacts with HPF1 the FRET signal can be monitored using a common plate reader fluorimeter.We validated that the signal results from the specific interaction by competing it out with unlabeled HPF1.The signal and interaction in nanomolar concentrations is sensitive to salt making it convenient to use just high NaCl concentration for the control in lack of known inhibitors.Extensive efforts in optimization resulted in an assay which showed consistently a stable and robust signal (Z' > 0.7) and can be run with automation in low 10 µl volumes in 384well plates.The assay requires > 100 nM protein concentrations, but, as specialized reagents are not needed and the recombinant fusion proteins are produced in E. coli, the assay is cost effective and can be applied to large screening campaigns.Recently, there was a study to find inhibitors against HPF1-PARP1 complex, where the interference of compounds between HPF1 and PARP1 was indirectly monitored by measuring the retention time of PARP1 on DNA 36 .This fluorescence polarization assay also identified catalytic PARP inhibitors and therefore the mechanism of the identified compounds may not be evident.In contrast, in the FRET-assay we describe here we measure directly the interaction between HPF1 and PARP and the hits, if not interfering with the signal or destabilizing proteins like crystal violet, are expected to act as protein-protein interaction inhibitors.The assay  www.nature.com/scientificreports/works even in the presence of a catalytic clinically used inhibitor and therefore selects the inhibitors mechanistically and avoids compounds only binding to the catalytic site unless the hit compounds extend from the catalytic site to the HPF1 binding interface.This feature extends the utility of the assay to test the mechanism of inhibitors identified by other means 36,37 .
During the validatory screening we discovered two hit compounds Dimethylacrylshikonin and Alkannin.These compounds stabilized PARP2 indicating direct binding and had µM potencies for both PARP1-HPF1 and PARP2-HPF1 interaction.The inhibitor compounds were also tested against full length PARP1/2 activity in presence of HPF1 and they were showing inhibitory effect on PARP activity.The screening method will hopefully facilitate the discovery of potent inhibitors against HPF1-PARP1/2 complexes and help to develop effective therapeutic molecules for cancer with a unique mechanism.

Protein expression
Plasmid containing CFP-PARP2 and YFP-HPF1 were transformed into E. coli BL21(DE3) and CFP-PARP1 was transformed to E. coli Rosetta2 (DE3) cells for the protein expression and incubated at 37 °C overnight.Transformed colonies were further inoculated in 500 ml autoinduction media which contains trace elements and supplied with 0.8% (w/v) glycerol, 50 μg/ml kanamycin antibiotic.PARP inhibitors 10 mM Benzamide and 2 mM 3-Amino Benzamide were added to the culture medium of CFP-PARP2 and CFP-PARP1, respectively 42 .Cultures were incubated at 200 rpm at 37 °C until an OD600 of 1.2, and then incubated for 18 h at 16 °C.Next day, cells were harvested by centrifugation at 4200 × g for 45 min at 4 °C.Pellets were resuspended in lysis buffer (50 mM HEPES, 350 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, 10% (w/v) glycerol, pH 7.5) and 100 µM pefablock [4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride] protease inhibitor was added in it.Resuspended cells were flash frozen in liquid nitrogen and stored at − 20 °C.

FRET measurement
FRET signal measurements were performed using Tecan Spark multimode plate reader.Measurement was done using 384-well plate (proxiplate-384 F plus from Perkin Elmer).CFP was excited using monochromator at 410 nm (20 nm bandwidth) excitation wavelength, emission was recorded at emission wavelengths of 477 nm (10 nm bandwidth) and 527 nm (10 nm bandwidth).Other parameters such as number of flashes 50, Z position 20,500, signal integration time 40 µs, settle time 10 ms, manual gain 90 were optimized and used for the FRET measurement.To calculate the ratiometric FRET (rFRET) value, fluorescence intensity at 527 nm was divided by fluorescence intensity at 477 nm.All the rFRET measurements were done in optimized buffer 10 mM Bis-Tris-Propane, 0.5 mM TCEP, 3% (w/v) PEG 20k, and 0.01% (v/v) TritonX-100, pH 7.5, unless otherwise stated in negative control samples, 1 M NaCl was added.To validate the inhibition of FRET pair by hit compounds, the 50% inhibition concentration (IC 50 ) values inhibitors against PARP1/2-HPF1 interaction, were measured using dilution series (100 µM to 3.25 nM) of compounds.Data was plotted against log inhibitor concentrations vs rFRET signal, analyzed in GraphPad Prism 9.0 using nonlinear regression analysis and IC 50 values for compounds were calculated.

PARP1/2 activity assay on SDS-PAGE
PARP1/2 activities were performed on SDS-PAGE gel as described by Langelier et al. 34 using the reaction buffer containing 50 mM Tris, 5 mM MgCl 2 (10 × concentration), pH 8.0.For the inhibitory effect on the PARP1/2 activity, 1 µM PARP1 or 1 µM PARP was mixed with 1 µM central nicked double stranded dumbbell DNA (5'p GGG TCT TTT GAC CCT CGA GCT TTT GCT CGA 3') and incubated for 10 min at room temperature.1 µM HPF1 followed by 500 µM NAD + were added to the DNA protein mixture were incubated for 5 min.Reaction was stopped by adding 500 µM Olaparib to the reaction.Selected reactions were treated with 1 M hydroxylamine (NH 2 OH) for 1 h at room temperature.Inhibitory compounds were added to the buffer before starting the reaction.SDS loading buffer was added to the reactions, samples were heated at 95 °C and run on the 4-20% SDS-PAGE gel stained subsequently with page blue staining solution.

Figure 4 .
Figure 4. CFP-PARP2 and YFP-HPF1 FRET pair signal competition with unlabeled HPF1 and determination of a dissociation constant.(a) FRET pair competition with unlabeled HPF1 containing salt (black) and equivalent salt (25 nM-350 mM; grey).(b) FRET pair competition with desalted unlabeled HPF1.(c) FRETbased determination (Kd) for CFP-PARP2 with YFP-HPF1 interaction.The FRET fluorescence emissions (Em FRET ) were determined as described by Song et al. 31 .All the reactions were performed in 20 µl volume in 384 well plates and the data shown are mean with standard deviations of 4 replicates.

Figure 6 .
Figure 6.rFRET signal for CFP-PARP1 and YFP-HPF1 FRET pair.(a) rFRET signal from control ( −) and FRET pair ( +) at different protein concentrations of CFP-PARP1 and YFP-HPF1.(b) Time dependent monitoring of rFRET signal, with 200 nM CFP-PARP1 and 400 nM YFP-HPF1 protein concentration.All the reactions were performed in 10 µl volume in 384 well plates and the data shown is mean ± standard deviation with 4 replicates.